Single cell transcriptomics unveiled that early life BDE-99 exposure reprogrammed the gut-liver axis to promote a pro-inflammatory metabolic signature in male mice at late adulthood (Part 2/2)
Data files
Apr 24, 2024 version files 193.18 GB
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CV1_CV_vs_GF.fastq.gz
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CV2_CV_vs_GF.fastq.gz
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CV3_CV_vs_GF.fastq.gz
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GF1_CV_vs_GF.fastq.gz
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GF2_CV_vs_GF.fastq.gz
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GF3_CV_vs_GF.fastq.gz
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README.md
Abstract
Polybrominated diphenyl ethers (PBDEs) are legacy flame retardants that bioaccumulate in the environment. The gut microbiome is an important regulator of liver functions including xenobiotic biotransformation and immune regulation. We recently showed that neonatal exposure to BDE-99, a human breast milk-enriched PBDE congener, up-regulated pro-inflammation- and down-regulated drug metabolism-related genes predominantly in males in young adulthood. However, it remains unknown regarding the persistence of dysregulation into late adulthood, differential impact of hepatic cell types, and the involvement of the gut microbiome from neonatal BDE-99 exposure. To address these knowledge gaps, male C57BL/6 mouse pups were orally exposed to corn oil (10 ml/kg) or BDE-99 (57 mg/kg) once daily from postnatal days 2-4. At 15 months of age, neonatal BDE-99 exposure down-regulated key xenobiotic- and lipid metabolizing enzymes and up-regulated genes involved in microbial influx in hepatocytes. Neonatal BDE-99 exposure also persistently increased the hepatic proportion of neutrophils and led to a predicted increase of macrophage migration inhibitory factor signaling. This was associated with decreased intestinal tight junction protein (Tjp) transcripts, persistent dysbiosis, and dysregulation of inflammation-related metabolites. ScRNA-seq using germ-free (GF) mice demonstrated the necessity of a normal gut microbiome in maintaining hepatic immunotolerance. Microbiota transplant to GF mice using large intestinal microbiome from adults neonatally exposed to BDE-99 down-regulated Tjp transcripts and up-regulated several cytokines in the large intestine. In conclusion, neonatal BDE-99 exposure reprogrammed cell type-specific gene expression and communication in liver towards pro-inflammation, and BDE-99-mediated pro-inflammatory signatures may be partly due to the dysregulated gut environment.
README: Single cell transcriptomics unveiled that early life BDE-99 exposure reprogrammed the gut-liver axis to promote a pro-inflammatory metabolic signature in male mice at late adulthood (Part 2/2)
Description of the data and file structure
The repository contains part 1 of 2 data of mouse single cell RNA sequencing fastq files which can be processed using the cell ranger pipeline (10X Genomics).
Part 1 contains 6 samples comparing the cell type-specific gene expression profiles using adult mouse livers neonatally exposed to BDE-99: DOI: 10.5061/dryad.4mw6m90gm
Part 2 (this dataset) contains 6 samples to investigate the necessity of the microbiome in regulating the hepatic transcriptome in mice: DOI: 10.5061/dryad.8931zcrzx
The Dryad file upload contains the raw single cell RNA sequencing (scRNA-seq) data comparing conventional (i.e., with microbiome) and germ-free (i.e., without microbiome) wild type (C57BL/6) mice. Mice were 7-8 month old and scRNA-seq was performed using dissociated livers. Fastq files containing CV1, CV2, and CV3, and GF1, GF2, and GF3 represent conventional and germ-free mouse livers, respectively.
Methods
Chemicals and dosing regimen
2,2’,4,4’,5-pentabromodiphenyl ether (BDE-99 [CAS No. 60348-60-9]) was purchased from AccuStandard, Inc. (New Haven, Connecticut). Corn oil was purchased from Sigma-Aldrich (St. Louis, MO). BDE-99 was dissolved in corn oil and filtered using a 0.22 μm Millipore Express Plus Membrane filter (EMD Millipore, Temecula, California). All mice used in this study were housed according to the Association for Assessment and Accreditation of Laboratory Animal Care International guidelines. The study was approved by the University of Washington Institutional Animal Care and Use Committee. Eight-week-old specific pathogen-free C57BL/6J mice were purchased from the Jackson Laboratory (Bay Harbor, Maine), and were acclimated to the animal housing facility at the University of Washington for at least three breeding generations. Mice were housed in standard air-filtered cages using autoclaved bedding (autoclaved Enrich-N’Pure, Andersons, Maumee, OH). Mice had ad libitum access to non-acidified autoclaved water, as well as standard rodent chow (LabDiet No. 5021 for breeding pairs or to LabDiet No. 5010 for weaned pups) (LabDiet, St. Louis, MO). From postnatal day (PND) 2 to PND 4, male pups were supralingually exposed to BDE-99 (57 mg/kg, n = 5) or corn oil (vehicle control, 10 ml/kg, n = 3) once daily for three consecutive days. Litters and cages were randomly assigned to each exposure group. Pups were weaned at PND 21. At 15 months of age, serum, liver, small and large intestines, and fresh stool were collected. The remaining livers were subject to dissociation. To determine the role of the microbiome in regulating hepatic cell types, livers from 7~8-month-old conventional (CV) or germ-free (GF) mice were collected.
Whole liver dissociation
Fresh whole livers rinsed in Dulbecco's phosphate-buffered saline (DPBS) (Cat# 14190136, Thermo Fisher Scientific, Waltham, MA) were minced to 1 – 3 mm pieces using surgical scissors. The minced livers were then placed in 15 mL conical tubes containing 10 mL of DPBS. Using serological pipettes, the DPBS was replaced by 10 mL of dissociation enzyme mixtures containing Liberase (Cat# 540115001, Sigma Aldrich, St. Louis, MO) and Dispase II (Cat# D4693-1G, Sigma Aldrich, St. Louis, MO) dissolved in Hanks' Balanced Salt Solution (Cat# 14025-092, Thermo Fisher Scientific, Waltham, MA). The tubes with the livers and enzyme mix were incubated at 37 °C for 40 minutes using a Roto-Therm H2020 incubator (Benchmark Scientific Inc., Sayreville, NJ). After the incubation, the cells and un-dissociated fragments were strained using a 40-micron cell strainer. Strained cells were centrifuged at 300 g for 5 minutes at 4 °C and re-suspended in 5 mL red blood cell lysis buffer on ice for 8 minutes. Cells were centrifuged again at 300 g for 5 minutes at 4 °C. Dead and dying cells were filtered using the Dead Cell Removal Kit (Miltenyi Biotec, Cologne, Germany) following the manufacturer’s instructions. The viability was checked using a hemocytometer under a light microscope (Labophot-2, Nikon, Tokyo, Japan). Samples with greater than 85% viability were then cryopreserved using 10% Dimethylsulfoxide (Cat# BP231-100, Thermo Fisher Scientific, Waltham, MA) and 90% fetal bovine serum (Cat# F2442-50ML, Sigma Aldrich, St. Louis, MO) until further analysis.
Single cell RNA sequencing
Cryopreserved cells were thawed using a water bath at 37 °C for 2 minutes, followed by serial dilution in DPBS until 32 mL was reached. Cells were centrifuged and re-suspended in DPBS until a concentration of approximately 100 cells/ μL was reached. The re-suspended cells (n=3), targeting 10,000 cells per sample, were then subject to scRNA-seq using a Chromium Next GEM single cell 3’ v3.1 kit and a Chromium X controller (10X Genomics, Pleasanton, CA) following the manufacturer’s instructions. The created libraries were then sequenced using the NovaSeq platform at paired-end 150 bp (~11 M reads per sample).
Data analysis of single cell RNA sequencing
Raw data were processed using the Cell Ranger v7.0 (10X Genomics, Pleasanton, CA). Processed data were read into R version 4.2.2 (R Core Team, 2022) for further analyses. Filtering and normalization were performed using the default parameters using Seurat v4 (Hao et al., 2021). Clustering was performed using the first 35 principal components, and the standard deviation obtained through principal component analysis was 1.5. Cell type labeling was performed using the differentially expressed genes using the function FindAllMarkers with default parameters in Seurat. Cell-cell communication analysis was done using the CellChat (v. 1.5) package (Jin, 2022). Gene ontology enrichment was done using the TopGO (v.2.48.0) package (Alexa A, 2022) using all detected genes as the background. Heatmaps were made using the ComplexHeatmaps (v. 2.13.1) package. All plots other than heatmaps were created using ggplot2 (v. 3.3.6).
Liver Hematoxylin and Eosin staining
A fraction of the liver from each mouse was preserved in 4% formalin (Cat# SF100-4, Thermo Fisher Scientific, Waltham, MA) followed by 70% ethanol for histology. Hematoxylin and Eosin (H&E) staining was performed by the University of Washington Histology Core in the Department of Comparative Medicine. Briefly, all staining procedures were performed in a well-ventilated area with personal protective equipment. Liver slides were prepared, dried overnight, and placed in the oven at 60˚C for approximately 30 minutes. The slides were stained in the following protocol: xylene for 5 minutes, rinsed in 100% ethanol for 4 minutes, 95% ethanol for 2 minutes, de-ionized (DI) water for 1 minute, hematoxylin for 3.5 minutes, DI water for 1 minute, clarifier for 30 seconds, DI water for 1 minute, bluing for 1 minute, DI water for 1 minute, eosin for 15 seconds, 95% ethanol for 1.5 minutes, 100% ethanol for 2 minutes, and xylene for 4 minutes. Images were then taken (Nanozoomer HT-9600, Hamamatsu Photonics, Japan), and histological incidence and severity scoring was performed by a board-certified veterinary pathologist.
Metagenomic shotgun sequencing
DNA from large intestinal content was extracted using the EZNA Stool DNA kit (Omega Bio-Tek Inc., Norcross, GA). Shallow shotgun metagenomic sequencing was performed at 2 million reads (Diversigen, New Brighton, MN). DNA sequences were aligned to a curated database containing all representative genomes in RefSeq for bacteria with additional manually curated mouse-specific Metagenomically Assembled Genomes (MAGs) and cell-cultured genomes. Only high-quality MAGs (Completeness > 90% & Contamination < 5% via checkm) were considered. Alignments were made at 97% identity against all reference genomes. Every input sequence was compared to every reference sequence in the Diversigen DivDB-Mouse database using fully gapped alignment with BURST. Ties were broken by minimizing the overall number of unique Operational Taxonomic Units (OTUs). For taxonomy assignment, each input sequence was assigned the lowest common ancestor that was consistent across at least 80% of all reference sequences tied for best hit. Taxonomies are based on Genome Taxonomy Database (GTDB r95). Samples with fewer than 10,000 sequences were discarded. OTUs accounting for less than one-millionth of all strain-level markers and those with less than 0.01% of their unique genome regions covered (and < 0.1% of the whole genome) at the species level were discarded.
For functional analysis, Kyoto Encyclopedia of Genes and Genomes Orthology groups (KEGG KOs) were used with alignment at 97% identity against a gene database derived from the strain database used above (DivDB-Mouse). KOs were collapsed to level-2 (phylum) and -3 (class) KEGG pathways and KEGG Modules. Count tables for taxonomic, enzyme, and pathway data were transformed using the centered-log ratio (CLR) method (Quinn et al., 2019). Statistical testing for all count data was analyzed using ANCOM-BC2 (Lin and Peddada, 2020) with BH-FDR < 0.05. Significant taxa were plotted as heatmaps using the R package ComplexHeatmap (v.2.13.1). Bar plots were created using ggplot2 (v 3.3.6).
Quantification of short-chain and medium-chain fatty acids
Short-chain and medium-chain fatty acids and their intermediate precursors were quantified as previously described (Gu et al., 2021; Dutta et al., 2022; Gomez et al., 2021). Briefly, approximately 50 mg of each tissue sample were homogenized in a mixture of 20 μl hexanoic acid-6,6,6-d3 (IS; 200 µM in H2O), 20 μl sodium hydroxide solution (NaOH, 0.5 M in water), and 480 μl methanol (MeOH). Then 400 μl of methanol was added. The pH of the mixture was adjusted to approximately 10. Samples were stored under −20°C for 20 min and then centrifuged at 21,694 × g for 10 minutes. A final volume of 800 μl of supernatant was collected. Samples were then evaporated to dryness, reconstituted in 40 μl of methoxyamine hydrochloride in pyridine (20 mg/ml), and stored at 60°C for 90 minutes. Subsequently, 60 μl of N-Methyl-N-tert-butyldimethylsilyltrifluoroacetamide was added and samples were heated to 60°C for 30 minutes. Samples were then vortexed for 30 seconds and centrifuged at 21,694 × g for 10 min. Finally, 70 μl of supernatant was collected from samples for gas chromatography-mass spectrometry (GC-MS) analysis.
GC-MS experiments were performed on an Agilent 88760 GC-5977B MSD system (Santa Clara, California) by injecting 1 µl of prepared samples. Helium was used as the carrier gas with a constant flow rate of 1.2 ml/min. The separation of metabolites was achieved using an Agilent HP-5ms capillary column (30 m × 250 × 0.25 µm). The column temperature was maintained at 60°C for 1 minute, and then increased at a rate of 10°C/minute to 325°C and held at this temperature for 10 min. The injector temperature was 250°C, and the operating temperatures for the transfer line, source, and quadruple were 290°C, 230°C, and 150°C, respectively. Mass spectral signals were recorded after a 4.9 min solvent delay. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for each metabolite in R for the analysis of liver metabolites (adjusted p-value < 0.05).
Fecal microbiota transplantation
To investigate the functional role of changes in microbial composition in the large intestine, fecal microbiota transplant (FMT) was administered to 8-12 week-old male germ-free adult mice using the large intestinal content of adult conventional mice that were neonatally exposed to vehicle or BDE-99. The FMT procedure was based on previous publications (Turnbaugh et al., 2006; Ridaura et al., 2013). The intestinal content of donors was flushed out of the large intestine with sterile PBS. The intestinal content was then diluted to approximately 50 mg/mL in sterile PBS. Each sample was mixed with a 1 mL pipette thoroughly. 200 μl of the diluted homogenate was orally gavaged to the germ-free recipients (n = 3-5). After one month of colonization, tissues were collected from the inoculated ex-germ-free mice and stored at -80 °C until further analysis.